Deep vein thrombosis is a common disease. Well established risk factors include recent surgery, malignant disorders, pregnancy and labour, long term immobilisation, and deficiency of one of the main inhibitors of the clotting system (Ref. 1). The main inhibitors are known to be protein C, protein S and antithrombin. The causes of deep vein thrombosis in many patients remain unclear. It has recently been established however that a poor anticoagulant response to activated protein C (APC) is present in several families with a hereditary tendency to venous thrombosis (Ref. 2).
The anticoagulant property of APC resides in its capacity to inactivate the activated cofactors Va and VIIIa by limited proteolysis (Ref. 3). This inactivation of cofactors Va and VIIIa results in reduction of the rate of formation of thrombin, the key enzyme of coagulation. Tn vitro, this effect can be visualised by adding APC to normal plasma and accordingly determining the effect thereof in a coagulation test, for example in a test determining the APTT ( activated partial thromboplastin time). Activation of protein C occurs at the surface of endothelial cells via the thrombin-thrombomodulin complex (Ref. 27). Thrombomodulin is a membrane glycoprotein that can bind thrombin. By this binding thrombin loses the ability to convert fibrinogen to fibrin and the ability to activate blood platelets. In other words thrombin loses its coagulant properties and reduces its further own production (so-called negative feed-back) by activating protein C. In vivo (in the presence of calcium) the activation of protein C is almost completely dependent on the availability of thrombomodulin on the endothelium. APC is subsequently neutralized by formation of complexes with APC inhibitor (PCI) and .varies..sub.1 antitrypsin, which means that in normal conditions it remains only for a short time in the circulation and the anticoagulant effect remains generally locally expressed.
It was generally accepted that the inactivation of the cofactors Va and VIIIa by APC proceeds only optimally in the presence of Ca.sup.2 +, phospholipids and the APC cofactor protein S (Ref. 4,28,29). More recently this view was, however, challenged by the finding that in systems of purified proteins protein S has little cofactor activity to APC (Ref. 5, Ref. 6). A possible solution for this apparent discrepancy between the observations in vivo (thrombotic tendency in hereditary protein S deficiency) and in vitro (poor APC cofactor activity of protein S in systems of purified proteins) could be offered by the finding of Dahlback et al (Ref. 2) who reported patients with normal values for antithrombin activity, protein C (immunologically and functionally) and protein S (immunologically and functionally) without indications for abnormal plasminogen, abnormal fibrinogen or lupus anticoagulants, but with a reduced anticoagulant response to activated protein C. The latter was found with a new test developed by Dahlback (Ref. 2) in which he studies the response (coagulation time, APTT) of a plasma after in vitro addition of purified human APC. The addition of activated protein C to the plasma of these thrombotic patients did not result in the expected prolongation of the activated partial thromboplastin time (APTT). After postulating a number of mechanisms for this phenomenon only one was considered to provoke the poor anticoagulant response to APC, namely the existence of a hitherto unknown cofactor to APC that is deficient in these patients.
The following mechanisms have to date been rejected as being causes of the poor anticoagulant response to APC:
1. The presence of an auto antibody against APC PA1 2. A fast acting protease inhibitor reacting with APC PA1 3. A functional protein S deficiency PA1 4. Mutations in the Factor V or Factor VIII gene
Dahlback (Ref. 2,7) postulated that in the families studied a hereditary shortage of a hereto unknown APC cofactor that purportedly works independently of protein S was the cause of APC resistance. Dahlback et al (Ref. 2) also described a test method for diagnosing the thrombo embolic disorders by addition of activated protein C to a patient sample containing coagulation factors followed by measurement of an enzyme activity that is influenced by the addition of APC in an international patent application wO93/10261. It is stated in the application of Dahlback that the experimental results presented indicated that the disorders in question are related to a hitherto unknown coagulation factor or factors or unknown interactions of known factors. The unknown factor is not Factor Va or VIIa that is resistant to degradation by APC and neither is it an inhibitor of the immunoglobulin type for APC. Furthermore it is not related to protein S deficiency. Dahlback et al (Ref. 2) state that their invention is a method particularly useful for further diagnosis of throtboembolic diseases such as hereditary or non hereditary thrombophilia and for determining a risk for thrombosis in connection with pregnancy, taking anticonception pills, surgery etc. They describe their method as being characterized in that the coagulation system in a sample is activated, wholly or partly in a manner known per se and incubated with activated protein C, whereupon a substrate conversion reaction rate like clotting or conversion of a chromogenic substrate is determined. The conversion rate obtained is compared with values obtain for normal plasma samples. If the rate is enhanced it indicates that the individual farm which the sample is derived may suffer from a clotting disease. The disease is not expressed by protein S deficiency or by formation of Factor Va or Factor VIIIa resistant to degradation by APC or by an inhibitor of the imunoglobulin type for APC. In the international application it is also stated by Dahlback et al that the data in the application indicated that the patient in question could not carry a detective Factor VIII/VIIIa in contrast to what they had previously stated in Thromb. Haemostas. 65, Abstract 39. 658 (1991), wherein addition of activated protein C to a plasma sample of a patient, and study of the effect produced was claimed to have illustrated a defective Factor VIIIa molecule not degraded by activated protein C. Furthermore in the international patent application the assay was used to directly measure the inhibition of Factors Va and VIIIa by APC. Using the Factor Xa based clotting assay described therein the inhibition of patient Factor Va by APC was found to be normal suggesting that Factor Va in the patient's plasma was degraded in a normal fashion by exogeneously added APC.
Following the publication by Dahlback et al (Ref. 2) other groups started research in this area. In Blood Vol. 82, nr. 7, 1993 on pages 1989-1993 Griffin et al describe the results of APC resistance tests carried out among 25 venous thrombotic patients with no identifiable blood coaglation abnormality and 22 patients previously identified with heterozygous protein C or protein S deficiency. The APC induced prolongation of the activated partial thromboplastin time assay for these patients was compared with results for 35 normal subjects. The results showed that this new defect in anticoagulant response to APC was surprisingly present in 52 to 64% or the 25 patients i.e. in the majority of previously undiagnosed thrombophilia cases. The deficiency was not present in 20 of 22 heterozygous protein C or protein S deficient patients. This suggested that the new factor is a risk factor independent of protein C or protein S deficiency. Mixing of normal blood plasma with each of two extremely defective plasmas (APC-induced prolongation of APTT &lt;20 s) was performed and the APTT assays were made to assess the ability or normal plasma to correct the poor response of the defective plasma. The results were similar to those of Dahlback et,al (Ref. 2). This also suggested that normal plasma contains a factor which is missing from the defective patients plasma. Values are given in the article for the net calculated prolongation in APTT. simply defined as an APTT value in the presence of APC minus the APTT value in the absence of APC. The article also describes the ratio of the APTT with APC to the APTT without APC and the fact that this parameter was compared to values for the APC induced APTT prolongation. This comparison indicated an excellent correlation between these parameters for the normals, with an extremely low APTT ratio value being indicative of abnormal patients. Therefore it followed that either the parameter of APC-induced ATT prolongation or the parameter of the ratio of APTT values with versus without APC or both parameters can be used as diagnostic parameter. None of these parameters seemed more useful for this purpose than the other according to the article. Furthermore in the article it was stated that the APC-induced prolongation of the APTT assay used was reminiscent of the assay involving APC-induced inactivation of endogenous Factor VIII in the plasmas of patients with lupus anticoagulant reported by Potzsch et al (Ref. 19) in Blood 80: 267a 1992 (Abstract)). Based on this latter assay it was reported in the Griffin et al article that plasma from lupus anticoagulant patients with thrombosis gave a poor response to APC and that patients with thrombosis could thereby be distinguished from those without thrombosis. Griffin et al speculated that auto-antibodies against the new hypothesized APC-cofactor could play a role in the risk of thrombosis among patients with lupus anticoagulants. It is further stated by them that it is tempting to speculate that an acquired deficiency of the new APC-cofactor could be associated with an acquired risk of thrombosis.
In the Lancet, Dec. 18, 1993, Vol. 342, on pages 1503-1506 Koster et al have elaborated further on the link between APC-resistance and thrombosis by describing how a population based case control study was undertaken to test the clinical importance of the abnormality in the coagulation system that is characterized by a poor anticoagulent response to activated protein C (AP). From studies within families this poor response to APC appears to inherit as an autosomal dominant trait (Ref. 2, 7 and 47). Among patients referred to a coaglation unit because of unexplained thrombosis this abnormality was a major cause of thrombophilia with a prevalence of about 40% (Ref. 8 and 9). In the study described by Koster et al in the Lancet, Dec. 18, 1993. Vol. 342, pages 1503-1506, the clinical importance of this poor response to APC was investigated in unselected consecutive patients aged less than 70 years, with a first objectively confirmed episode of deep vein thrombosis and without an underlying malignancy. The sensitivity of these patients plasma to APC was compared with that of matched healthy controls. The sensitivity of their plasma APTT to activated Protein C (APC) was measured essentially as described by Dahlback et al (Ref. 2) using the reagents and reaction conditions previously developed for the protein S activity assay (Ref. 11). The results were empresses as APC sensitivity ratios (APC-SR) defined as the value of APTT (+APC) over the APTT (-APC). In the Koster et al article (Lancet. Dec. 18, 1993, Vol. 342, pages 1503-1506) it was stated that reduced levels of prothrombin and/or Factor X (&lt;0.5 u/ml) will increase the APC-SR. For this reason the test cannot be used for the evaluation of plasma's of patients on oral anticoagulant treatment. In a series of 98 samples a good correlation was found between the APC-SR obtained with the test of Koster et al (Lancet, Dec. 18, 1993, Vol. 342, pages 1503-1506) and those obtained with the test developed by Chromogenix as described in WO 93/10261. A reference range for the APC sensitivity ratio was derived from the healthy control subjects. After logarithmic transformation of the data and exclusion of 10 subjects with values outside 3 standard deviations (SD) of the mean, the lower limit of normal was 2.17 (mean-1.96 SD). An inverse relation between the risk of thrombosis and the degree of response was found. The 21% prevalence of a poor response to APC among thrombosis patients and the odds ratio for thrombosis of 6.6 led to the conclusion that a poor response to APC could be considered a common and strong risk factor for deep vein thrombosis. It was even speculated that subjects with APC sensitivity ratios around 1.10 could be homozygous or double heterozygous, whereas subjects with APC sensitivity ratios around 1.50 could be heterozygous for the abnormality. The prevalence of the abnormality was 5% among the healthy control subjects. Because the distribution of the APC-SR was clearly bimodal Koster et al believe that subjects really had abnormal responses to APC rather than too low values within a normal range. The relation between risk of thrombosis and their response to APC seemed therefore not to follow the model of a simple single gene defect. Because the abnormality was found to be so prevalent in healthy subjects it was considered unlikely by Koster et al that the defect in itself is sufficient to cause thrombosis as is true also for protein C deficiency (Ref. 15, Ref. 16). An additional causal factor seems to be required for the development of thrombosis within a particular patient. These may be acquired factors and also as yet unknown genetic defects or variations. However once other causal factors are present poor APC response presents a strong risk of thrombosis as witnessed by its six to sevenfold increase of relative risk. It was stated in the article that the underlying defect of the poor response to APC remained to be clarified even though a dominantly autosomally inherited deficiency of a cofactor to activated protein C had been postulated (Ref. 7). While a poor response to APC appears to be 5 to 10 times as frequent as deficiencies of protein C, protein S or antithrombin it confers an approximately similar relative risk of thrombosis (Ref. 17 and 18) which according to Koster et al could make it worthwhile to test all patients with venous thrombosis for this abnormality.
In summary in the state of the art it was ascertained that a defect in the protein C anticoagulant pathway is linked to a relatively high risk of thrombosis. The poor anticoagulant response to activated protein C has been discussed in great detail, however the cause of the poor anticoagulant response to activated protein C remains unclear. A number of theories have been postulated, however the only one that has been accepted is the presence of an unknown cofactor for APC which is apparently deficient in a patient exhibiting a poor anticoagulant response to activated protein C. The identity of the postulated cofactor for APC is unknown. Furthermore current tests for detecting altered response to APC cannot be used on test persons already using anticoagulants.